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WO1995018379A1 - Methode d'analyse d'un complexe forme entre la secretine et un ligand de celle-ci et utilisation dudit complexe - Google Patents

Methode d'analyse d'un complexe forme entre la secretine et un ligand de celle-ci et utilisation dudit complexe Download PDF

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Publication number
WO1995018379A1
WO1995018379A1 PCT/JP1994/002203 JP9402203W WO9518379A1 WO 1995018379 A1 WO1995018379 A1 WO 1995018379A1 JP 9402203 W JP9402203 W JP 9402203W WO 9518379 A1 WO9518379 A1 WO 9518379A1
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WIPO (PCT)
Prior art keywords
antibody
selectin
ligand
complex
recognizes
Prior art date
Application number
PCT/JP1994/002203
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English (en)
Japanese (ja)
Inventor
Reiji Kannagi
Naofumi Takahashi
Miki Kitade
Naoko Hashimoto
Hitomi Mimuro
Original Assignee
The Nisshin Oil Mills, Ltd.
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Publication date
Application filed by The Nisshin Oil Mills, Ltd. filed Critical The Nisshin Oil Mills, Ltd.
Priority to AU12806/95A priority Critical patent/AU1280695A/en
Publication of WO1995018379A1 publication Critical patent/WO1995018379A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/645—Secretins

Definitions

  • the present invention relates to a method for measuring a complex of selectin and a ligand thereof and a kit for diagnosing vascular-related diseases such as cancer metastasis, inflammatory disease or diabetes. More specifically, the present invention relates to an antibody that recognizes the selectin portion of a complex of selectin and its ligand, and an antibody that recognizes the ligand portion. A qualitative and / or quantitative assay for the complex, and cancer metastasis comprising a set of an antibody recognizing the selectin portion of the complex of selectin and its ligand and an antibody recognizing the ligand portion, The present invention relates to a kit for diagnosing vascular diseases such as inflammatory diseases or diabetes. Background art
  • Selectin is one of a family of intercellular adhesion molecules, and three molecules, L-selectin, P-selectin and E-selectin, have been discovered. Each of these molecules is a type I cell membrane glycoprotein with an N-terminus outside the cell and a C-terminus inside the cell. From the N-terminus toward the cell membrane, lectin domain, EGF Gene cloning has revealed that it has three domains, called domains and a complement-binding domain that repeats sequences found in complement-binding proteins.
  • L-selectin is a molecule that is expressed on leukocytes and is responsible for the phenomenon of lymphocyte migration to specific locations, particularly the transition from the vascular system to peripheral lymph node homing (lymphocyte homing). When they adhere to vascular endothelial cells, they make a special interaction called leukocyte rolling. E-selectin is expressed on activated vascular endothelial cells and is involved in the binding of neutrophils, monocytes, memory T cells, eosinophils and basophils to vascular endothelial cells. P-selectin is expressed on activated platelets and activated vascular endothelial cells and is involved in the binding of platelets to neutrophils or monocytes and the binding of neutrophils to activated vascular endothelial cells.
  • L-selectin is expressed without stimulation in almost all leukocytes, and E-selectin and P-selectin are induced by inflammatory sites such as IL-11 ⁇ TNF ⁇ . It is clear that it will be.
  • ⁇ -selectin is also expressed by histamine and thrombin.
  • soluble selectin in serum meaning selectin released from vascular endothelial cells, platelets or leukocytes
  • E-selectin has high specificity for vascular endothelial cells and is therefore used for diagnosis of vascular-related diseases.
  • 2-3 Shiariru L e x and 2-3 Shiariru L e * is used as a tumor marker since ancient times molecule having a sugar chain structure
  • 2-3 Shiariru L e x in serum 2- Antigens expressed by cancer cells, such as 3 sialyl Le 4 have been used for cancer diagnosis.
  • Shiariru L e x is, 2 ⁇ 3 Shiariru L e x antigen that specifically recognizes monoclonal anti Washed After reacting with the addition of serum or standard containing 2 ⁇ 3 Xia Lil L e x antigen to the body to immobilize the beads or 96 blade bets Ueru, then Peruokishidaze or aralkyl force Li off O and labels with a radioactive isotope of the enzyme or 1 2 5 I such as scan Fataze 2 ⁇ * 3 Shiariru L e x antigen were washed after specifically reacted with the addition of recognizing labeled mono clonal antibody , then either measured by color development the absorbance was added to specific substrates in enzyme, or by measuring the levels of the direct radiation is the method expressed as 2 ⁇ 3 Shiari Le L e x antigen amount .
  • Cancer metastasis can be attributed to (1) the escape of cancer cells grown in the primary focus and their migration into blood vessels, (2) the migration of cancer cells into blood vessels, (3) the adhesion of cancer cells to peripheral vascular endothelium, and (4) the basement membrane of cancer cells. It is thought to be established through four stages: the formation of metastatic lesions by infiltration into connective tissue. Thus, adhesion of cancer cells to vascular endothelial cells is considered to be one step in the hematopoietic transition of cancer.Recently, a cell adhesion molecule of the selectin family and cancer cells are expressed.
  • Glucose-mediated adhesion such as 2 ⁇ 3 sialyl Le x and 2-3 sialyl Le ′, has been shown to play an important role in this step. Therefore, close involvement of selectins in cancer metastasis has been pointed out.
  • Cancer is a disease in which some cells begin to grow abnormally. They invade surrounding normal tissues and continue to grow, destroying normal functions. Furthermore, cancer cells have the ability to metastasize, which is one of the reasons that cancer is also called a malignant tumor. The ability of cancer cells to metastasize varies depending on the type of cancer that arises, with some cancer cells having a strong metastatic capacity and others having poor metastatic capacity. Therefore, it is difficult to predict the possibility of cancer metastasis by measuring each tumor marker in serum, although it is possible to know the progress of cancer. Therefore, a diagnostic method that can predict the possibility of cancer metastasis is desired.
  • the present inventors have conducted intensive studies on more accurate methods of diagnosing cancer metastasis and diagnosing inflammatory diseases and vascular-related diseases.
  • vascular endothelial cells, platelets and leukocytes are strongly activated, inner skin cells, selectin expressed on platelets and white blood cells are liberated, from the cancer cells and leukocytes, were expressed in cancer cells and leukocytes 2 ⁇ 3 Xia Lil L e x or 2 ⁇ 3 Xia Lil L e * like Na selecti And ⁇ the ligand of emissions, liberated selectin and its ligand forms a complex in the blood, by the child measuring the formed complex, selectins and 2 ⁇ 3 Shiariru L e x or 2-3 Find out that cancer metastasis, inflammatory disease or vascular disease can be diagnosed more accurately than measuring a selectin ligand such as sialyl Le * alone, and complete the present invention. Reached.
  • an antibody recognizing a selectin portion of a complex of selectin and its ligand is brought into contact with a biological sample, and an antibody recognizing the ligand portion is brought into contact with the biological sample.
  • a method for qualitatively and Z- or quantitatively measuring a complex of selectin and its ligand comprising measuring the binding between an antibody that recognizes the selectin portion and an antibody that recognizes the ligand portion of the complex.
  • the present invention comprises a set of an antibody recognizing a selectin portion of a complex of selectin and a ligand thereof and an antibody recognizing the ligand portion, including metastasis, inflammatory disease or diabetes.
  • the present invention provides a diagnostic kit for vascular-related diseases.
  • the biological sample used in the present invention whole liquid components obtained from the inside of a living body can be applied, and examples thereof include serum, plasma, lysate, urine, synovial fluid, nasal secretion, and cerebrospinal fluid.
  • serum and plasma it is preferable to use serum and plasma as the biological sample.
  • the method for measuring the complex of selectin and its ligand according to the present invention is based on all selectin molecular species expressed in vascular endothelial cells, platelets, leukocytes, and the like, and all selectin molecular species expressed in cancer cells, leukocytes, and the like. It can be applied to the measurement of all kinds of complexes formed from the molecular species.
  • Is a selectin molecule species forming a complex for example E- selectin, P- selectin, L Serekuchi emissions and the like, is a ligand species of selectin, For example 2-3 Shiariru L e x , 2-3 as an example of a complex of Shiari selectin Le L e 'or the like is measured by the recited process of the present invention and its re cancer de, for example E- selectin and 2 ⁇ 3 Xia Li Le L e x complex, E- selectin and 2 ⁇ 3 complex with Xia Li Le L e ', P- selectin and complex with 2 ⁇ 3 Xia Li Le L e x with, P - selectin and 2 ⁇ 3 Xia Lil complex with L e *, a complex with L Sere cutin and 2 ⁇ 3 Xia Lil L e x, complexes such as the L over selectin and 2 ⁇ 3 Xia Lil L e a can be mentioned.
  • measuring method in the present invention is particularly applicable to the measurement of E- selectin and 2 ⁇ 3 complex with Shiariru L e x and E- selectin and 2 ⁇ 3 Xia Lil L e * and the complex It is preferable to be done.
  • selectin refers to vascular endothelial cells such as E-selectin, P-selectin and L-selectin, platelets, leukocytes, etc. It is to be understood that this encompasses all selectin species that are expressed by, and also simply referred to as “selectin ligand J", but “selectin ligand” refers to 2-3 cyaryl L it should be understood to include all Li cancer de species for e chi and 2 ⁇ 3 Shiariru L e 'cancer cells and leukocytes, etc. Thus the expressed selectins such.
  • ⁇ complex J of selectin and its ligand '' is simply referred to as ⁇ complex of selectin and its ligand ''.
  • the leukocytes include, for example, neutrophils, eosinophils, basophils, monocytes, and lymphocytes, and in the following description of the specification, the force simply referred to as "white blood cells”. It should be understood that eosinophils, basophils, monocytes and lymphocytes are included.
  • the antibody that recognizes the selectin portion of the complex of selectin and its ligand used in the present invention may be any antibody that recognizes any region as long as it reacts with the selectin in a state of being adhered to the ligand.
  • the antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Further, these antibodies may be fragments such as F (ab ') 2 and Fab in addition to the complete molecule.
  • These antibodies can be prepared, for example, by using soluble E-selectin, P-selectin or L-selectin as an antigen to prepare a polyclonal antibody or a monoclonal antibody against the molecular species, and producing the antibody.
  • cells such as vascular endothelial cells (expressing selectin) activated by a cytokine that induces the expression of selectins such as IL-11 / S can be used as antigens for polyclonal antibodies.
  • Antibody that recognizes the desired selectin species from the produced antibody, and recognizes the selectin moiety adhered to the ligand from the separated antibody. can be produced by separating the two.
  • the ELAM-1 (synonymous with E-selectin) fragment up to the 420th amino terminal is produced by genetic engineering. This fragment contains the lectin domain, the EGF domain, and a portion of the complement fixation domain. Glut this fragment Rabbits are immunized multiple times by binding to KLH (keyhole limpet hemocyanin), an immunoadjuvant, by the Lualdehyde method. Polyclonal antibodies can be obtained by obtaining antiserum from the rabbit after immunization, and then purifying the antiserum by the ammonium sulfate method and DEAE anion column chromatography.o
  • Examples of the method for preparing a monoclonal antibody against selectin used in the present invention include, for example, Bevilacqua et al. [Bevillacqua et al., Pro Natl. Acad. Sci. 84: 9238-9242 (1987)] and Voter et al. The method reported by Porter et al., J. Immunol. 136: 1680 (1986)], and the mouse hybridoma method described in W0 / 9 005539 and W0 / 9005786 can be applied. The specific method is described below. The case where vascular endothelial cells activated by a cytokine that induces the expression of selectin such as IL-11 as an antigen will be described as an example.
  • mice are immunized by intraperitoneal administration of vascular endothelial cells (expressing selectin) activated by IL-11 ⁇ to mice, and then spleen cells are obtained from the mice.
  • the immunized spleen cells are then subjected to myeloma using polyethylene glycol according to, for example, the method of Kohler and Milstein (Kohler, Milstein, Nature. 256: 495-497 (1975)). Fuse with cells.
  • fused cells that react with IL-11-activated vascular endothelial cells but do not react with normal resting vascular endothelial cells (hereinafter, the fused cells are called hybridomas) are subjected to limiting dilution. To clone.
  • the ELAM-1 antibody (for example, ELAM-1 antibody BBA-2 manufactured by Pretty Biotechnology Inc.) can be immobilized on a 96-well plate, and the ELAM-1 antibody can be used.
  • a normal human umbilical cord-derived vascular endothelial cell lysate containing the above is added and reacted to wash and remove unreacted components, and then the prepared polyclonal antibody or monoclonal antibody is added, for example, after labeling with an enzyme. The unreacted components are washed away. Then, a specific substrate is added to the enzyme to develop color and the absorbance is measured.
  • Antibodies to ELAM-1 can be separated based on the obtained absorbance.
  • IL-11 normal human umbilical cord-derived vascular endothelial cells cultured in a 24-well plate until they are completely dense are stimulated with a cytokine such as IL-11, and the supernatant is removed.
  • the prepared polyclonal antibody or monoclonal antibody is added to the IL-11 / 5-treated group and allowed to react (IL-11-treated + antibody-treated group).
  • IL-11-treated + antibody-treated group As controls, an IL-1; 5 untreated group (IL-11 untreated group) and an IL-11y5 treated group (IL-11 ⁇ -treated group) to which the prepared antibody was not added were prepared.
  • a cancer cell line expressing a selectin ligand encapsulating a fluorescent dye in each treatment group [eg, a human colon cancer cell line CO L020 (from Dainippon Pharma Co., Ltd.) can be applied and centrifuged. Wash each well, add 0.5% Triton X-100, stir, and dispense it into a 96-well plate to measure the fluorescence intensity. From the results of the fluorescence intensities measured in the IL-1 ⁇ 8 untreated group, the IL-11 treated group, and the IL-1 ⁇ treated + antibody treated group,
  • Adhesion inhibition rate (%) 100— ((Fluorescence intensity of group treated with IL-1 ⁇ + antibody) 1 (Fluorescence intensity of group not treated with 1L-1 / S) Z (Fluorescence intensity of group treated with IL-1yS (Fluorescence intensity) 1 (1 L-1 1 Untreated group fluorescence intensity)] X 100
  • the adhesion inhibition rate () is determined using. Based on the obtained adhesion inhibition rate (%), an antibody that recognizes the selectin portion of the complex of selectin and its ligand can be separated. The antibody thus obtained recognizes the selectin moiety in a state of being adhered to the ligand.
  • an antibody that recognizes the selectin portion of the complex of selectin and its ligand prepared as described above can be used, and the known selectin of the complex of selectin and its ligand can be used.
  • An antibody that recognizes the portion can also be used.
  • known antibodies include, for example, the monoclonal antibody BBIG-E5 against ELAM-1 described in The Journal of Immunology (The Journal of Immunology: Vol. 147, Vol. 130-135. No. 1, July 1 1991), and a polyclonal antibody against ELAM-1 reported by Newman et al. ELAM-1 420 (Newman, Double et al., J. Immunol. 150: 64 4-654 (1993)].
  • the antibody used in the present invention that recognizes the ligand portion of the complex may be a polyclonal antibody or a monoclonal antibody as long as it is an antibody against a selectin ligand species, but it is a monoclonal antibody. Is preferred.
  • polyclonal antibodies or monoclonal antibodies that recognize the ligand portion of the complex of selectin and its ligand can be used as a cancer cell or leukocyte cell expressing a selectin ligand as an antigen. It can also be prepared by using such cells. In that case, it is necessary to separate an antibody recognizing the desired ligand portion of the complex from the produced antibody.
  • cells such as vascular endothelial cells are used as antigens.
  • a method according to a method for separating an antibody recognizing a selectin portion of a desired complex from the produced antibody can be applied.
  • Many of the selectin ligands exist in a living body in a polyvalent state (a state in which a large number of sugar chains having antigen epitopes are bound to a core protein) in an antibody that recognizes the ligand portion of the complex. Therefore, the antibody may be an antibody that recognizes a region involved in selectin adhesion, or may be an antibody that recognizes a region not involved in selectin adhesion.
  • the antibody that recognizes the ligand portion of the complex includes a selectin ligand or a ligand portion prepared by using a cell such as a cancer cell or a leukocyte cell expressing the selectin ligand as an antigen.
  • an antibody that reacts with a selectin ligand can also be applied.
  • examples of such an antibody include, for example, an antibody against a known selectin ligand [for example, CSLEX 1 (Chia, D .; (Chia, D.) et al., Cancer Res. 45: 435-437 (1985) 3. These include FC ab 'as well as intact molecular antibodies, as well as antibodies that recognize the selectin portion of the complex. ) Use fragments such as 2 and Fab
  • the method for measuring the complex of selectin and its ligand generally includes either an antibody that recognizes the selectin portion of the complex of selectin and its ligand or an antibody that recognizes the ligand portion. After immobilizing one of them as a first antibody on a support (or immobilizing it), a sample containing a complex of selectin and its ligand (biological test) ) To cause an antigen-antibody reaction (first reaction), wash and remove unreacted sample components, and then recognize an antibody or ligand that recognizes the selectin portion of the complex of selectin and its ligand.
  • the second antibody which is not the antibody used as the first antibody, is reacted as the second antibody (second reaction), and the second antibody is labeled with a labeling compound to form the second antibody.
  • Second reaction a labeling compound to form the second antibody.
  • Figure 1 shows an example in which the first antibody immobilized on the support is an antibody that recognizes the selectin portion of the complex between selectin and its ligand. The principle of the law is shown.
  • the first antibody immobilized on the support may be either an antibody recognizing the selectin portion of the complex of selectin and its ligand or an antibody recognizing the ligand portion, but an antibody recognizing the selectin portion may be used. preferable.
  • a support generally used in this field can be applied.
  • polystyrene beads, tubes, A micro evening plate, a strip and the like can be mentioned, and among them, a micro evening plate is preferable.
  • the second antibody of the antibody recognizing the selectin portion or the antibody recognizing the ligand portion of the complex, the other, which is not the antibody used as the first antibody, is used.
  • an antibody that recognizes a ligand portion is preferable.
  • the second antibody is preferably labeled with a labeling compound, and as the labeling compound for labeling the second antibody, a labeling compound generally used in the art can be used. Enzymes, coenzymes, fluorescent dyes, radioisotopes and the like are mentioned, and enzymes are preferred. If an unlabeled second antibody is used, use an appropriate reagent (for example, if the second antibody is a mouse IgM monoclonal antibody, use an anti-mouse Ig IV [antibody]). Can be detected.
  • the second antibody When an enzyme, a fluorescent dye or a radioisotope is used as a labeling compound for labeling the second antibody, the second antibody can be directly labeled with them, and their enzymatic activity and fluorescence in the reacted second antibody can be labeled. By measuring the intensity or radioactivity, the binding of the complex of the formed first antibody-selectin and its ligand to the second antibody can be measured.
  • the second antibody is labeled with a coenzyme, and the binding substance that binds to the coenzyme is labeled with an enzyme, a fluorescent dye, or a radioisotope to supplement the coenzyme.
  • Enzymes and anti By reacting (binding) and measuring the enzymatic activity, fluorescence intensity or radioactivity of the labeled compound in the binding substance bound to the coenzyme.
  • Preferred examples of the enzyme used as the labeling compound include, for example, peroxidase, alkaline phosphatase, ⁇ -galactosidase, galactose dehydrogenase, lingoic acid dehydrogenase, acetylcholinesterase, glucose oxidase and the like. Of these, peroxidase is particularly preferred.
  • a method for measuring the enzyme activity of these enzymes a method generally used in the art can be applied, and examples thereof include a colorimetric method, a fluorescent method, a bioluminescent method, and a chemiluminescent method. .
  • a colorimetric method for example, when peroxidase (hereinafter, abbreviated as D) is used as an enzyme, orthophenylenediamine (hereinafter, abbreviated as OPD) or 3, 3 ', 5, 5'- A colorimetric method using tetramethylbenzine or the like as a substrate specific to the enzyme (hereinafter abbreviated as a substrate), a fluorescence method using 4-hydroxyphenylacetic acid or 3- (4-hydroxyphenyl) propionic acid as a substrate, or Lumi no Lou Eta 2 0 2 may be to measure the enzyme activity by chemiluminescence method or the like is used as a substrate.
  • alkaline phosphatase When alkaline phosphatase is used as the enzyme, 412 trophenyl phosphatase or Enzyme activity can be measured by a colorimetric method using NADP or the like as a substrate, or a fluorescence method using 4-methylbenveriferyl phosphate or the like as a substrate.
  • yS-D-galactosidase When yS-D-galactosidase is used as the enzyme, a colorimetric method using 2-nitrotropinyl ⁇ -D-galactoside as a substrate, 4-methyl umbenylfuryl-1D-galactoside, etc.
  • the enzyme activity can be measured by a fluorescence method using chromium as a substrate, or a chemiluminescence method using 2-2-trophinyl-D-galactoside or the like as a substrate.
  • a fluorescence method using chromium or a chemiluminescence method using 2-2-trophinyl-D-galactoside or the like as a substrate.
  • the enzyme activity can be measured by a bioluminescence method or the like using glucose 16-phosphate or the like as a substrate, and glucose oxidase is used as the enzyme.
  • enzymatic activity can be measured by a chemiluminescence method or the like using peroxyxarate or the like as a substrate.
  • fluorescent dye used as a labeling compound include, for example, fluorescein isothiosinate (FITC), tetramethylrhodamine inthiosinate (RITC:), phycoerythrin (PE), Texas red (TE), And arophycocyanine (APC).
  • FITC fluorescein isothiosinate
  • RITC tetramethylrhodamine inthiosinate
  • PE phycoerythrin
  • TE Texas red
  • APC arophycocyanine
  • radioisotope used as a labeling compound, for example, 125 1, 131 3 H, 19 C , or the like. When these radioisotopes are used, their radioactivity can be measured directly.
  • the capture enzyme used as the labeling compound include, for example, biotin, and examples of the binding substance that binds to biotin include, for example, streptavidin and avidin.
  • Preferred examples of the dye or radioisotope are the same as described above.
  • a method for directly labeling the second antibody with an enzyme, a fluorescent dye, or a radioisotope a method generally used in the relevant field can be applied, for example, a glutaraldehyde method, periodate, and the like. Method, maleimide method, pyridyl disulfide method and the like.
  • a method of labeling the second antibody with a coenzyme for example, a method according to the method of labeling with biotin described in “Immunological Experiment Procedures, edited by the Immunology Society of Japan, p. 3830” can be applied. The specific method is as follows.
  • Antibodies were dissolved in 0.1 MN a HC 0 3 to a concentration of 1 mgZinl, dissolving NHS- Piochin 1 mg to DMS 0 of 1 ml.
  • To 1 ml of the above antibody solution add 601 of NHS-Piotin dissolved in DMSO, react at room temperature for 4 hours, and then dialyz well with phosphate-buffered saline (hereinafter abbreviated as PBS). By doing so, a biotinylated antibody can be obtained.
  • PBS phosphate-buffered saline
  • avidin labeled with POD may be one manufactured by Vector.
  • the fragment when a fragment such as F (ab ') 2 or Fab is used as the second antibody in addition to the complete molecule, the fragment can be labeled by the method described above.
  • the enzyme activity of P ⁇ D in avidin conjugated with biotin is determined using a biotinylated antibody as the second antibody and avidin labeled with P 0 D as the substance that binds to biotin. It is most preferable to measure the binding between the formed first antibody-selectin and the complex of the ligand and the second antibody.
  • the concentration of the first antibody to be added to the support is preferably 50 g / ml, and the solvent used for diluting the first antibody is generally used in the art.
  • PBS carbonate buffer, Tris-HCl buffer and the like can be used, and PBS is preferable.
  • the amount of the first antibody to be added to the support is appropriately 25/1 to 250 1, preferably 50 1 to 100 1.
  • After adding the first body to the support leave at 4-37 ° C, preferably 4 ° C, for 10-72 hours, preferably 12-24 hours. This allows the first antibody to be immobilized on the support.
  • the first antibody may be fixed to an amino- or carboxyl-group-modified support.
  • the blocking agent used for such blocking a blocking agent generally used in the relevant field can be applied, and examples thereof include a 1% BSA solution and normal rabbit herb serum.
  • Commercially available products include Block Ace manufactured by Dainippon Pharmaceutical.
  • the blocking agent is preferably used by adding sodium azide as a preservative at a concentration of 0.05%.
  • the appropriate addition amount of the blocking agent is 251 to 250 ⁇ 1, and 200 ⁇ 1 is preferred.
  • the first antibody on the support is immobilized by leaving the plate at 4-37, preferably at 4 ° C, for 10-30 hours, preferably 12-48 hours. You can block the missing parts.
  • a detergent used for washing a detergent generally used in the relevant field can be applied, for example, 0.1% BSA-PBS, 0.1% normal persimmon serum-PBS, 10% block Ace One PBS It is preferable to use 10% Block Ace-PBS.
  • the number of washings is appropriately 1 to 5 times, preferably 2 to 3 times.
  • a sample biological sample
  • the amount of addition is suitably from 25 to 250 to 1, preferably from 501 to 1001.
  • the addition of the sample causes an antigen-antibody reaction (first reaction) between the first antibody and the complex of selectin and its ligand.
  • the reaction temperature of the first reaction is suitably from 4 to 37 ° C, preferably from 20 to 25 ° C.
  • the reaction time is suitably 30 minutes to 24 hours, preferably 1 to 2 hours.
  • a second antibody (labeled antibody) is added.
  • concentration of the second antibody to be added is preferably 2 ⁇ g / ml.
  • solvents for diluting the second antibody Is the same as the example of the solvent for diluting the first antibody.
  • the addition amount of the second antibody is appropriately from 25 1 to 250; 1, and preferably from 50 1 to 100 1.
  • an antigen-antibody reaction (second reaction) between the second antibody and the complex of selectin and its ligand occurs.
  • the appropriate reaction temperature and reaction time for the second reaction The same is true. That is, the reaction temperature is suitably from 4 to 37 ° C., preferably from 20 to 25, and the reaction time is suitably from 30 minutes to 24 hours, preferably from 1 to 2 hours.
  • the formed first antibody-selectin and its reactant are determined by measuring the enzyme activity, fluorescence intensity or radioactivity of the labeled compound in the reacted second antibody. Complex with ligand The binding of the primary antibody can be measured.
  • the unreacted second antibody is removed by washing, and a binding substance (labeled with a labeling compound) that binds to the coenzyme is added to the second antibody. It is necessary to react (coupling) with the coenzyme in the antibody.
  • concentration of the binding substance to be added is appropriately 0.5 to 12 g / ml, preferably 2 to 6 ⁇ g / ml, and the addition amount is preferably 25 1 to 250 1, and 50/1 to 50 g / ml. 100 ⁇ 1 is preferred.
  • the reaction temperature is suitably 4 to 37 ° C, preferably 20 to 25 ° C, and the reaction time is suitably 10 to 120 minutes, preferably 20 to 60 minutes.
  • the enzyme activity, fluorescence intensity or radioactivity of the labeled compound in the reacted label-binding substance is measured to determine the relationship between the formed first antibody-selectin and its ligand.
  • the binding of the complex-second antibody can be measured.
  • the unreacted second antibody or the labeled binding substance is washed away, and then reacted with a substrate specific to the enzyme by a method common in the art.
  • the enzyme activity can be measured.
  • the amount of the substrate to be added is preferably 50 to 1001.
  • the reaction temperature is 4 to 37 e C is suitable, preferably 15 to 25 ° C, the reaction time is suitably 5 to 60 minutes, preferably 1 0 to 20 min.
  • a reaction terminator generally used in this field can be applied, and examples thereof include a sulfuric acid solution.
  • the addition amount of the reaction terminator is preferably from 50 to 1001. After stopping the reaction, the enzyme activity can be measured by measuring the desired absorbance.
  • the complex of selectin and its ligand can be measured qualitatively and z- or quantitatively. That is, based on the measured enzymatic activity, fluorescence intensity or radioactivity, the formation of a complex between the first antibody-selectin and its ligand-the formation of the second antibody's bond was confirmed, whereby the selectin and its ligand were determined. Can be confirmed (qualitative measurement) by using a dilution series of a standard substance (a standard substance of a complex of selectin and its ligand) instead of a biological sample. Of the complex between the selectin and its ligand (quantitative) by measuring the binding of the second antibody to the complex of the first antibody-selectin and its ligand formed by the reaction Measurement) can be measured.
  • the first antibody is immobilized on a support, then blocked and washed, to obtain a first antibody-immobilized plate.
  • a sample containing a complex of selectin and its ligand biological sample
  • a second antibody concentration and addition amount are as described above
  • a binding substance that further binds to the coenzyme (the (Concentration and amount of addition are as described above), and leave to react for 4 to 37, preferably 20 to 25, 30 minutes to 24 hours, preferably 1 to 2 hours.
  • an appropriate amount of the mixture in a test tube is added to the first antibody-immobilized plate, and the mixture is left for 4 to 37, preferably 20 to 25, for 30 minutes to 24 hours, preferably for 1 to 2 hours.
  • the enzyme activity, fluorescence intensity or radioactivity of the labeled compound is measured to bind the formed first antibody-selectin and its complex with the ligand-second antibody. Can be measured.
  • the measurement method of the present invention has good daily variation, daily variation, and simultaneous reproducibility, and can measure the amount of a complex of selectin and its ligand with good reproducibility.
  • the method for measuring a complex of selectin and its ligand according to the present invention is useful for diagnosis of vascular-related diseases such as cancer metastasis, inflammatory disease or diabetes.
  • the diagnosis of blood vessel-related diseases such as metastasis, inflammatory disease or diabetes is based on confirming the presence or absence of a complex of selectin and its ligand in a biological sample. However, if the presence of a complex of selectin and its ligand is confirmed, it is possible that inflammatory diseases or vascular-related diseases such as diabetes may be occurring. This indicates that metastases may have occurred. Also raw If a complex of selectin and its ligand in a body sample is quantitatively measured and a high value is obtained, it is possible that cancer metastasis or vascular disease such as inflammatory disease or diabetes is occurring. It is shown to be high.
  • cancer metastasis and inflammatory disease comprising a set of an antibody recognizing a selectin portion of a complex of selectin and its ligand and an antibody recognizing the ligand portion are provided.
  • a diagnostic kit for a disease or a vascular-related disease such as diabetes is provided.
  • the antibody that recognizes the ligand portion of the complex between selectin and its ligand in the diagnostic kit of the present invention is preferably labeled with a labeled compound.
  • Examples of the labeled compound are as described above. The same is true.
  • the diagnostic kit of the present invention is a diagnostic kit for carrying out the method for measuring the complex of selectin and its ligand according to the present invention, and the measuring method is described above.
  • the diagnostic kit according to the present invention contains reagents, supports, standard substances, and the like necessary for carrying out the above-described measurement method, and these reagents and standard substances are stored in a closed container. It is preferably stored.
  • the diagnostic kit of the present invention should be stored in a cool place. If the diagnostic kit of the present invention is stored at 10 or less, it is stable for a long time. Yes, 30. It is not preferable to store at C or more, since the antibody activity decreases. Therefore, the storage temperature of the diagnostic kit of the present invention is preferably 4 to 25 ° C, and 4 to 10. C is preferred. It is not preferable to store the diagnostic kit of the present invention for 13 months or more, since the activity of the antibody decreases. Therefore, about one year is appropriate for the storage period of the diagnostic kit of the present invention.
  • Biological samples used for diagnosis are not preferred because storage at 0 or lower makes the antigen stable, and storage at 1 or higher makes the antigen unstable. Therefore, the storage temperature of a biological sample, one 80 to 0 e C are suitable, arbitrary favored -80 one 20 ° C is. In addition, if the biological sample is stored for more than 13 months, the antigen will be unstable, so a storage period of about 1 year is appropriate for the biological sample. However, if it is necessary to store the biological sample for a long time before the diagnosis is made, storing it at a temperature of -80 or lower will not significantly reduce the reliability of the diagnosis result. Will not.
  • the method for measuring the complex of selectin and its ligand according to the present invention is useful for diagnosis of vascular-related diseases such as cancer metastasis, inflammatory disease or diabetes.
  • vascular-related diseases such as cancer metastasis, inflammatory disease or diabetes.
  • kidney cancer, 2 ⁇ 3 Shiariru L e x is predominantly expressed in adenocarcinoma cells, such as lung and breast cancer, ⁇ , cancer and adenocarcinoma of colon cancer such as biliary tree
  • the cancer cells 2 ⁇ 3 Shiariru L e * has summer so obvious that it is predominantly expressed, also 2 ⁇ 3 Xia Lil L e x or 2-3 Shiariru L e ⁇ expression in squamous carcinoma cells It is clear that this is being done.
  • Shiariru L e x and 2 ⁇ 3 Shiari Le L e * is believed to be involved in adhesion between vascular endothelial cells expressing cancer cells and selectins. Therefore, they strongly expressing cancer cells Shasuku Oko hematogenous transitional, in cancer cells, 2 - 3 When Shiariru L e 'or 2 ⁇ 3 Shiariru L e 11 is expressed more strongly, they from a cancer cell It is considered to be released into body fluids (especially in the blood, which will be described below in the blood).
  • E-selectin and P-selectin are expressed in vascular endothelial cells, and when vascular endothelial cells are strongly activated, their expression levels increase, and when E-selectin and P-selectin are more strongly expressed. It is thought that those selectins are also released from vascular endothelial cells into the blood. The released ligand and E-selectin or P-selectin form a complex in blood, and when this complex is measured, both cancer cells and vascular endothelial cells are activated. Thus, the possibility of activated cancer cells adhering (metastatic) to activated vascular endothelial cells can be diagnosed.
  • P-selectin Expression of P-selectin which is expressed not only in cells but also in platelets, and expressed in platelets, increases with the activation of platelets, and is considered to be released from platelets into the blood when more strongly expressed .
  • platelets and ⁇ metastasis if activated platelets adhere to cancer cells released into the blood, platelets are likely to be implanted on vascular endothelial cells and metastasis is likely to be established due to the inherently high adhesion of platelets. Can be Therefore, the possibility of cancer metastasis can be diagnosed by measuring the complex of P-selectin and its ligand in blood.
  • L-selectin is expressed in leukocytes, and is thought to similarly enter the blood by activating leukocytes.
  • the relationship between leukocytes and cancer metastasis is thought to be that when leukocytes adhere to cancer cells that have migrated into the blood, they adhere to vascular endothelial cells due to the ability of leukocytes to adhere and promote metastasis.
  • some leukemia and malignant lymphoma cells express L-selectin, in which case they adhere to the ligand sugar chains expressed in vascular endothelial cells, causing the invasion of these malignant cells into organs. It is thought to affect the pattern. Therefore, the possibility of cancer metastasis can be diagnosed by measuring the complex of L-selectin and its ligand in blood.
  • vascular related diseases including inflammatory diseases and diabetes, 2 ⁇ 3 Xia neutrophils with Li Le L e x as a surface antigen, monocytes and Note rie T cells subgroups [God ⁇ et al, Pathophysiology 7: 888-900 (198 8); MedicaIl Immunol., Knnnag i. 25: 89-95 (1993)] is likely to accumulate at the site of inflammation and progress by infiltrating into tissues.
  • selectins are more strongly expressed, they are likewise released into the blood ⁇ neutrophils, monocytes and memory-type also in T cell subgroups, 2 by which they are more strongly activated ⁇ 3 Shiariru L e x is believed that free in blood. Therefore, by measuring the complex of selectin and its ligand in blood, it is thought that vascular diseases such as inflammatory diseases and diabetes can be more accurately diagnosed.
  • FIG. 1 is a diagram showing the principle of the measuring method according to the present invention.
  • FIG. 2 is a diagram showing a specific example of the measuring method according to the present invention. The symbols in the figure indicate the following.
  • Normal human umbilical cord-derived vascular endothelial cells purchased from Kurabo Industries were added. The cells were cultured in an EGM-UV medium attached thereto, and 200 UZnil of recombinant IL-1 ⁇ (manufactured by Zenzym) was added thereto, and activated for 4 hours.
  • BALB / C mice were intraperitoneally administered with 3 ⁇ 10 6 IL-11 activated vascular endothelial cells three times every 10 days, and the last administration was performed 3 days before cell fusion.
  • the immunized splenocytes are fused with the P3U1 myeloma cell line using polyethylene glycol according to the method of Keller and Milstein (Kohler, Milstein. Nature.
  • Block Ace (Dainippon) (Manufactured by Pharmaceutical Co., Ltd.)-200% of -0.05% NaN3 solution was added thereto, and the mixture was left at 4 for 10 minutes.
  • Block Ace-PBS add 50 1 of normal human umbilical cord-derived vascular endothelial cell lysate containing ELAM-1 prepared as follows to each well, and add at room temperature. Left for 1 hour.
  • OPD O le Tofuwe two Renjiami emissions prepared by solution each 50 ⁇ 1 added was allowed to stand 10 minutes at room temperature, 50 1 added 2 MH 2 S 0 4 solution to each Ueru, The absorbance was measured at a wavelength of 492 nm. The absorbance obtained when the antibody obtained in 1 above was reacted with PBS was used as a control (blank). Table 1 shows the results.
  • Normal human umbilical cord-derived endothelial cell lysates containing ELAM-1 It was prepared as follows. Normal human umbilical cord-derived vascular endothelial cells (derived from Kurabo Industries) 3 ⁇ 10 6 cultured in a 150 cm 2 dish were treated with recombinant IL-11 (Zenzym) at 200 U / ml. C was stimulated for 4 hours. After the supernatant was aspirated and washed twice with PBS, 10 ml of a 1% Triton X-100-PBS solution was added, followed by pipetting. After collecting the suspended cell lysate, the supernatant was concentrated to 1 ml by centrifugation at lOOOOg for 2 hours, and the sample was used as 20000 UZml.
  • the biotinylated antibody was prepared according to the method described in “Immunological Experiment Procedures, edited by the Immunological Society of Japan, p. 3830.” First, the antibody was dissolved in 0.1 M NaHC03 to a concentration of l mgZ ml, 1 mg of S-Piotin (manufactured by Vector) was dissolved in 1 ml of DMS I. Next, 60 ml of NHS monoiotin dissolved in DMS O was added to 1 ml of the antibody solution, and then After reacting for hours, the mixture was dialyzed sufficiently against PBS to obtain a biotinylated antibody.
  • ⁇ PD solution is prepared by adding 20 ral of 0.1 MNa P04 to 1 tablet of OPD ⁇ (13 mg / l tablet, manufacturer: Wako Pure Chemical), adding 50% of 30% H 2 O 2 and stirring. Prepared.
  • the purified antibody at 20 g / ml was added to each of the rhIL-1 / 5 treated groups, and reacted at room temperature for 30 minutes (rhIL-15 treatment).
  • Treatment + antibody treatment group Thereafter, the human colon cancer cell line C0L0201 containing the fluorescent dye 2 ', 7'-bis (carboxicetyl) -1,5,6-carboxyfluorescein acetomethyl ester (hereinafter abbreviated as BCECF-AM) was introduced.
  • the cells were added at 1 ⁇ 10 6 cells / well and centrifuged at 100 rpm for 20 minutes.
  • Human colon cancer cell line C0L0201 encapsulating BCECF-AM was prepared by reacting BCECF-AM with cells.BCECF-AM enters the cell through the membrane and is degraded by intracellular esterase. It is present in human colon cancer cell lines as a membrane-impermeable BCECF.
  • Adhesion inhibition rate () 100 — ((rh IL-lg treated + antibody treated group fluorescence intensity) one (rh IL-1 ⁇ untreated group fluorescence intensity) / (rh IL-1; 5 treated group (Fluorescence intensity) 1 (rh1L-1; 5 Untreated group fluorescence intensity)] X100 Table 2
  • Table 2 indicates that No.93 is an antibody that recognizes the ELAM-1 portion of the complex of ELAM-1 and its ligand.
  • Each antibody was biotinylated by the method according to the method for producing a monoclonal antibody against biotinylated ELAM-1 in Example 1.
  • Piochin of 2 ⁇ 3 Shiariru L e x antibody 5A 12 were designated Piochin of 2 ⁇ 3 Shiariru L e 'antibodies No. 1 1 and.
  • Example 3
  • the ribosome dispersion is passed through a polycarbonate membrane filter (manufactured by Nuclepore, Inc.) having an average pore size of 0.2 ⁇ m so that the average particle size becomes 0.2 m, and the ribosome solution is added at 100 ⁇ g / ml. age
  • ELAM-1 antibody No. 93 50 g / ml diluted in PBS was added to each well of a 96-well plate, 50 ⁇ 1 each, and the antibody solution left at 4 ° C was collected.
  • Manufactured by Dainippon Pharmaceutical Co., Ltd. 1. 200 ⁇ l of a 0.05% NaN 3 solution was added to each well, and each well was left standing for 4 minutes. After washing each gel once with 10% Block Ace-PBS, normal human umbilical cord-derived vascular endothelial cell lysate ( ⁇ ) containing ELAM-1 was added to each gel 50/1/50 and added at room temperature. Left for hours.
  • ELA ⁇ - 1 antibody (No.93) one Piochin of 2 ⁇ 3 Xia Li Le L e x antibody (5A12) - avidinized P 0 D-color
  • EL AM - 1 antibody No.93
  • Normal human umbilical cord-derived vascular endothelial cells lysate containing EL AM- 1 - 2 ⁇ 3
  • Xia Li Le L e x Li Po Endosomal solution one Piochin of 2-3 Shiariru L e x antibody (5A12) - avidinated POD- color
  • ELAM-1 antibody No. 93 50 g / ml diluted with PBS was added to each well of a 96-well plate, 50 1 each, and the plate was allowed to stand at 4 ends for 1 minute. After recovering the antibody solution, block Kuesu one 0.05% N a N 3 solution was added to each well at 200 ⁇ 1 and left at 4 ⁇ for 1 ⁇ . After washing each well once with 10% Block Ace / PBS, add 50 ⁇ l of normal human umbilical cord-derived vascular endothelial cell lysate (5000 UZm 1) containing ELAM-1 to each well at room temperature. Left for 1 hour.
  • ELAM-1 antibody No. 93
  • ELAM-1 antibody No. 93
  • Sugar protein solution-biotinylated 2 ⁇ 3 sialyl Le ⁇ antibody No.11
  • mono-avidinated P OD color development
  • the ELMA-1 antibody did not react with the biotinylated 2 ⁇ 3 sialyl Le * antibody or the 2 ⁇ 3 sialyl Le ′ antibody, and the ELAM-1 and 2 ⁇ 3 sialyl L It is clear that e 'reacts only with the complex formed by binding.
  • ELAM-1 antibody No. 93 50 ⁇ g / ml diluted with PBS was added to each well of a 96-well plate at 50 1 and left at 4 ° C for 1 hour. After collecting the antibody solution, a block ace-0.05% NaN 3 solution was added to each well, 200 ⁇ 1 each, and left at 4 ° C for 1 ⁇ ⁇ . After washing each well once with 10% Block Ace-PBS, add the ELAM-1 Z2-3 Sialyl Le 'complex solution to each well.
  • Anti-2 ⁇ 3 sialyl Le 'antibody No. 11 (50 g / ml) diluted with PBS was added to each well of a 96-well plate, 50 1 each, and left at 4 ° C for 1 hour.
  • After recovering the antibody solution (manufactured by Dainippon Kusurisha) block Kuesu -0.05% N a N 3 solution 200 1 each added to each Ueru were 1 ⁇ left at 4 ° C.
  • Each well with 10% Block Ace-PBS After washing once, 2-3 1 sialyl Le 'glycoprotein solution (5000 U / ml) was added to each well, and left at room temperature for 2 hours.
  • Block Ace-PBS After washing each well three times with 10% Block Ace-PBS, add 50 ⁇ l of normal human umbilical cord-derived vascular endothelial cell lysate (5000 U / ml) containing ELAM-1 to each well, and add 2 wells at room temperature. Left for hours. After washing each well three times with 10% Block Ace-PBS, add 50 1 of bitionized ELAM-1 antibody No. 93 (2 fig / ml) to each well and leave at room temperature for 2 hours. did. After washing each well three times with 10% Block Ace-PBS, avidinated peroxidase (Vector; 2 ⁇ g / ml) was added to each well at 50 1 and left at room temperature for 30 minutes.
  • Vector 2 ⁇ g / ml
  • E LAM- 1 Z2- 3 Shiariru L e x complex preparation of the solution ELAM - 1 normally comprises a human umbilical cord-derived vascular endothelial cells lysate (10000 U / ml) and 2 ⁇ 3 Shiariru L e x ribosome (100 H g / ml) and then mixed with an equal volume sample was allowed to stand for 1 hour at room temperature, and E LAM- 1 Z2 ⁇ 3 Shiariru L e x complex solution was set to the concentration 10000 U / ml.
  • ELAM-1 antibody No. 93 50 ⁇ g / ml diluted with PBS was added to each well of a 96-well plate, 50 1 each, and left at 4 ° C for 1 hour.
  • block Kuesu one 0.05 ⁇ N a ⁇ 3 solution was added in portions 200 1 to each Ueru and 1 ⁇ left at 4 ° C.
  • Each gel was washed once with 10% Block Ace-PBS to obtain an ELAM-1 antibody-immobilized plate.
  • each of the serum 50 ⁇ to each test tube 1 and Piochin of 2 ⁇ 3 Shiariru L e x antibody CC sLeX-1] or Piochin of 2-3 Shiariru L e ' Add the antibody [2D3] (2 ⁇ g / ml) 50 / z1, leave at room temperature for 2 hours, add avidinated peroxidase (6 ⁇ g / l) 501 thereon, and add at room temperature. Left for 1 hour. This was added to 100 mL of the ELAM-1 antibody-immobilized plate and left at room temperature for 2 hours.
  • I is the measurement results of multiple polymer concentration of EL AM- 1 and 2 3 Shiariru L e x
  • I is EL AM- 1 and the complex concentration between 2 ⁇ 3 Shiariru L e lambda The measurement results are shown.
  • EL AM-1 2 ⁇ 3 Shiariru L e x with complex and ELAM 1 that there is a possibility that it is also monitored-ring from one of the complex of the 2 ⁇ 3 shea Ariru L e 'of, in gastric, ELAM 1 and by measurement of the complex of the 2 ⁇ 3 Shiariru L e x, considered can monitor-ring.
  • the concentration of the complex becomes high, so even in cancer patients at normal times (for example, before surgery), it is diagnosed that the higher the value, the higher the possibility of metastasis. can do.
  • the method for measuring a complex of selectin and its ligand according to the present invention is useful for diagnosis of vascular-related diseases such as cancer metastasis, inflammatory disease and diabetes.
  • vascular-related diseases such as cancer metastasis, inflammatory disease and diabetes.
  • 2 ⁇ 3 Shiariru L kidney expressing e x, colon cancer, lung cancer, ovarian cancer, Fugan, liver cancer, uterine cancer, breast cancer, leukemia and 2 ⁇ 3 Xia Li Le L e * ⁇ expressing It is useful for diagnosing metastasis in gastrointestinal cancers such as bile, liver, and colorectal cancers.

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Abstract

L'invention concerne une méthode d'analyse quantitative et/ou qualitative d'un complexe formé entre la sécrétine et un ligand de celle-ci qui consiste à mettre un échantillon biologique en contact avec un anticorps reconnaissant la fraction sécrétine du complexe, et un anticorps reconnaissant la fraction ligand de ce dernier, et à analyser la liaison du complexe avec l'anticorps reconnaissant la fraction sécrétine et avec l'anticorps reconnaissant la fraction ligand dans le produit de contact. L'invention porte également sur du matériel de diagnostic des métastases cancéreuses, de maladies inflammatoires ou vasculaires qui se compose d'un ensemble constitué de l'anticorps reconnaissant la fraction sécrétine et de l'anticorps reconnaissant la fraction ligand.
PCT/JP1994/002203 1993-12-27 1994-12-22 Methode d'analyse d'un complexe forme entre la secretine et un ligand de celle-ci et utilisation dudit complexe WO1995018379A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014098715A (ja) * 2014-02-12 2014-05-29 Denka Seiken Co Ltd 着色ラテックス粒子を用いるメンブレンアッセイ法およびキット

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5592693A (en) * 1978-09-21 1980-07-14 Hoechst Japan Kk Determination of ligand
WO1990005539A1 (fr) * 1988-11-14 1990-05-31 Brigham And Women's Hospital Anticorps specifiques contre elam-1 et leur utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5592693A (en) * 1978-09-21 1980-07-14 Hoechst Japan Kk Determination of ligand
WO1990005539A1 (fr) * 1988-11-14 1990-05-31 Brigham And Women's Hospital Anticorps specifiques contre elam-1 et leur utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BLOOD, Vol. 82, No. 9, (1993), OHMORI KATSUYUKI et al., "A Distinct Type of Sialy Lewis X Antigen Defined By a Novel Monoclonal Antibody is Selectively Expressed on Helper Memory T Cells", pages 2797-2805. *
JIKKEN IGAKU, Vol. 11, No. 16, (1993), KANNAGI REIJI et al., "Selectins and Cancer Metastasis", pages 2168-2175. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014098715A (ja) * 2014-02-12 2014-05-29 Denka Seiken Co Ltd 着色ラテックス粒子を用いるメンブレンアッセイ法およびキット

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